The effects of mutagens, such as radiation or chemotherapeutic agents, on the survival of spermatogenic cells of mouse testes and the damage produced in their DNA will be studied. Biochemical alterations in testicular cell DNA produced by mutagen treatment will be assayed in cells at each stage of spermatogenesis. The different stages can be studied by separation of cells and nuclei from the testis using velocity sedimentation and equilibrium density centrifugation. In addition, effects on cells at different stages may be studied by analyzing the DNA of sperm collected from the epididymis at various times after treatment. Direct damage to the DNA and repair will be assayed by measuring strand breaks, alkaline-labile bonds and endonuclease-sensitive sites by sucrose gradient centrifugation. Alterations of the DNA content of individual cells can be measured by flow microfluorometry. The restoration of fertility at long times after radiation is being compared to the regeneration of stem cells. We have developed an enzyme assay and a rapid microscopical method (counts of testicular sperm nuclei) to measure the survival of stem cell function, and are correlating these results with a histological method which is applicable only at high radiation doses. The enzyme assay and counts of sperm nuclei can detect effects of low doses. The ultimate objectives of this study are, using mice treated with radiation and mutagenic chemicals, to (1) understand the biochemical mechanisms underlying mutagenesis and, (2) quantitate survival of testicular stem cells and its relation to fertility in order to extrapolate these data to man. BIBLIOGRAPHIC REFERENCES: Meistrich, M. L., 1977. "Separation of Spermatogenic Cells and Nuclei from Rodent Testes." In Methods in Cell Biology, Vol. 15, D.M. Prescott, Ed., New York, New York, Academic Press, Inc. pp. 15-54. Platz, R.D., Grimes, S.R. and Meistrich, M.L., 1977. "Low Molecular Weight Basic Proteins in Spermatids." In Methods in Cell Biology, Vol. 16, D.M. Prescott, Ed., New York, New York, Academic Press, Inc. pp. 297-316 (in press).